Gas1 Mutant Research Articles

Molecular profiling of the vestibular lamina highlights a key role for Hedgehog signalling.

The vestibular lamina (VL) forms the oral vestibule, creating a gap between the teeth, lips and cheeks. In a number of ciliopathies, formation of the vestibule is defective, leading to the creation of multiple frenula. In contrast to the neighbouring dental lamina, which forms the teeth, little is known about the genes that pattern the VL. Here, we establish a molecular signature for the usually non-odontogenic VL in mice and highlight several genes and signalling pathways that may play a role in its development. For one of these, the Sonic hedgehog (Shh) pathway, we show that co-receptors Gas1, Cdon and Boc are highly expressed in the VL and act to enhance the Shh signal from the forming incisor region. In Gas1 mutant mice, expression of Gli1 was disrupted and the VL epithelium failed to extend due to a loss of proliferation. This defect was exacerbated in Boc/Gas1 double mutants and could be phenocopied using cyclopamine in culture. Signals from the forming teeth, therefore, control development of the VL, coordinating the development of the dentition and the oral cavity.

Gas1 Regulates Patterning of the Murine and Human Dentitions through Sonic Hedgehog

The mammalian dentition is a serially homogeneous structure that exhibits wide numerical and morphological variation among multiple different species. Patterning of the dentition is achieved through complex reiterative molecular signaling interactions that occur throughout the process of odontogenesis. The secreted signaling molecule Sonic hedgehog (Shh) plays a key role in this process, and the Shh coreceptor growth arrest-specific 1 (Gas1) is expressed in odontogenic mesenchyme and epithelium during multiple stages of tooth development. We show that mice engineered with Gas1 loss-of-function mutation have variation in number, morphology, and size of teeth within their molar dentition. Specifically, supernumerary teeth with variable morphology are present mesial to the first molar with high penetrance, while molar teeth are characterized by the presence of both additional and absent cusps, combined with reduced dimensions and exacerbated by the presence of a supernumerary tooth. We demonstrate that the supernumerary tooth in Gas1 mutant mice arises through proliferation and survival of vestigial tooth germs and that Gas1 function in cranial neural crest cells is essential for the regulation of tooth number, acting to restrict Wnt and downstream FGF signaling in odontogenic epithelium through facilitation of Shh signal transduction. Moreover, regulation of tooth number is independent of the additional Hedgehog coreceptors Cdon and Boc, which are also expressed in multiple regions of the developing tooth germ. Interestingly, further reduction of Hedgehog pathway activity in Shhtm6Amc hypomorphic mice leads to fusion of the molar field and reduced prevalence of supernumerary teeth in a Gas1 mutant background. Finally, we demonstrate defective coronal morphology and reduced coronal dimensions in the molar dentition of human subjects identified with pathogenic mutations in GAS1 and SHH/GAS1, suggesting that regulation of Hedgehog signaling through GAS1 is also essential for normal patterning of the human dentition.

GAS1: A New β-Glucan Immunostimulant Candidate to Increase Rainbow Trout (Oncorhynchus mykiss) Resistance to Bacterial Infections With Aeromonas salmonicida achromogenes.

β-glucans are prebiotic and/or food additives used by the aquaculture industry to enhance the immune response of fish. Their efficiency may vary according to their origin and structure. In this study, the immunostimulant effects of two β-glucan types extracted from wild-type baker’s yeast (Saccharomyces cerevisiae) and its null-mutant Gas1 were investigated. Gas1 has a beta-1,3-glucanosyltransferase activity necessary for cell wall assembly. Using a positive (commercial product MacroGard®) and a negative control (a diet without glucans), we evaluated the immune responses and disease resistance of rainbow trout juveniles (mean weight, ~44 g) fed control, low (0.2%) and high (0.5%) doses of Macrogard®, Gas1, and Wild type-β-glucan after a short-term (15 days, D15) or mid-term (36 days, D36) feeding periods. We found that β-glucan supplemented diets did not affect growth performance, mortality, splenic index, or leukocyte respiratory burst activity on D15 nor D36. However, each β-glucan triggered different immune effectors, depending of the doses or length of exposure compared to others and/or the negative control. Indeed, high dose of MacroGard® significantly increased lysozyme activities at D15 compared with the control and other diets (p<0.05). At D36, MacroGard β-glucan enhanced the production of lymphocytes in comparison with the control diet (p<0.05). Regarding WT β-glucan, at D36, WT-β-glucan, especially the high dose, provided the highest enzymatic activities (lysozyme and ACH50) and Ig level (p<0.01). Furthermore, on D36, Gas1 also increased lysozyme activity, Ig proportion, and some immune genes (mcsfra, hepcidin) compared with MacroGard® (p<0.05). Besides, both doses of Gas1-β-glucans increased the resistance of juveniles to bacterial infection highlighted by a higher survival rate at 14 days post-challenge compared with the control and other types and doses of β-glucans (p<0.05). In conclusion, our results suggest that Gas1-β-glucan could represent a promising immunostimulant that would help to prevent diseases in aquaculture even more efficiently than other β-glucans already in use. Mode of action and particular efficiency of this new Gas1 mutant are debated.

Investigating Novel Roles for Hedgehog Co‐Receptors During Craniofacial Development

Hedgehog (HH) signaling is essential for correct patterning of vertebrate embryos, including during craniofacial development. HH ligands signal through the canonical receptor Patched1 (PTCH1), and three co‐receptors–growth arrest specific 1 (GAS1), cell adhesion molecule‐related/down‐regulated by oncogenes (CDON), and brother of CDON (BOC). Together, these co‐receptors are required during embryogenesis to mediate proper HH signaling. However, recent genetic studies suggest tissue‐specific, antagonistic roles for these proteins. For example, Gas1 restricts the HH pathway during mouse tooth development, while Boc antagonizes HH signaling during zebrafish jaw formation. Here I explored possible antagonistic roles for GAS1, CDON and BOC in HH‐dependent mammalian craniofacial development. Genetic deletion of these co‐receptors results in variable holoprosencephaly (HPE) phenotypes (failure to divide the forebrain into two cerebral hemispheres). Gas1 mutant mice display a spectrum of HPE phenotypes, and significant patterning defects in the neural tube and limb. In contrast, genetic deletion of Boc does not perturb patterning in any of these tissues. Strikingly, the combined deletion of Boc and Gas1 results in more severe neural tube and limb patterning defects than those observed in Gas1 single mutants. However, the Gas1 craniofacial phenotype is partially ameliorated in Gas1;Boc double mutant embryos as determined by the levels of NKX2.1, Gli1, Ptch1 and internasal distance quantitation. Micro‐computed tomography and skeletal analyses reveal that the rescue of the craniofacial defects in Gas1;Boc double mutants is restricted to the nasal bone and nasal capsule; other craniofacial bones are more severely affected, similar to what is observed in the neural tube and the limb. This effect does not appear to be due to differential GAS1 and BOC function in the forebrain neuroepithelium as overexpression of these co‐receptors in the developing chicken forebrain both promote HH signaling. Instead, I hypothesize that these co‐receptors have distinct, tissue‐specific functions in the craniofacial mesenchyme or the surface ectoderm. To further explore an antagonistic role for BOC during craniofacial development, I am currently deleting Boc in a sensitized HH genetic background through breeding with Ptch1 and Shh heterozygous mutant animals. These findings suggest that GAS1 and BOC do not function simply as redundant HH co‐receptors, but instead display distinct, tissue‐specific functions, particularly during craniofacial development.Support or Funding InformationNational Sciences Foundation Graduate Research Fellowship Program (DGE 1256260), Rackham Pre‐Candidate Graduate Student Research Grant, Bradley Merill Patten Fellowship and Organogenesis Training Grant (NIH T32HD007505)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Sex and Mitonuclear Adaptation in Experimental Caenorhabditis elegans Populations

To reveal phenotypic and functional genomic patterns of mitonuclear adaptation, a laboratory adaptation study with Caenorhabditis elegans nematodes was conducted in which independently evolving lines were initiated from a low-fitness mitochondrial electron transport chain (ETC) mutant, gas-1 Following 60 generations of evolution in large population sizes with competition for food resources, two distinct classes of lines representing different degrees of adaptive response emerged: a low-fitness class that exhibited minimal or no improvement compared to the gas-1 mutant ancestor, and a high-fitness class containing lines that exhibited partial recovery of wild-type fitness. Many lines that achieved higher reproductive and competitive fitness levels were also noted to evolve high frequencies of males during the experiment, consistent with adaptation in these lines having been facilitated by outcrossing. Whole-genome sequencing and analysis revealed an enrichment of mutations in loci that occur in a gas-1-centric region of the C. elegans interactome and could be classified into a small number of functional genomic categories. A highly nonrandom pattern of mitochondrial DNA mutation was observed within high-fitness gas-1 lines, with parallel fixations of nonsynonymous base substitutions within genes encoding NADH dehydrogenase subunits I and VI. These mitochondrial gene products reside within ETC complex I alongside the nuclear-encoded GAS-1 protein, suggesting that rapid adaptation of select gas-1 recovery lines was driven by fixation of compensatory mitochondrial mutations.

Adaptive Evolution under Extreme Genetic Drift in Oxidatively Stressed Caenorhabditis elegans.

A mutation-accumulation (MA) experiment with Caenorhabditis elegans nematodes was conducted in which replicate, independently evolving lines were initiated from a low-fitness mitochondrial electron transport chain mutant, gas-1. The original intent of the study was to assess the effect of electron transport chain dysfunction involving elevated reactive oxygen species production on patterns of spontaneous germline mutation. In contrast to results of standard MA experiments, gas-1 MA lines evolved slightly higher mean fitness alongside reduced among-line genetic variance compared with their ancestor. Likewise, the gas-1 MA lines experienced partial recovery to wildtype reactive oxygen species levels. Whole-genome sequencing and analysis revealed that the molecular spectrum but not the overall rate of nuclear DNA mutation differed from wildtype patterns. Further analysis revealed an enrichment of mutations in loci that occur in a gas-1-centric region of the C. elegans interactome, and could be classified into a small number of functional-genomic categories. Characterization of a backcrossed four-mutation set isolated from one gas-1 MA line revealed this combination to be beneficial on both gas-1 mutant and wildtype genetic backgrounds. Our combined results suggest that selection favoring beneficial mutations can be powerful even under unfavorable population genetic conditions, and agree with fitness landscape theory predicting an inverse relationship between population fitness and the likelihood of adaptation.

Genetic interactions between the hedgehog co-receptors Gas1 and Boc regulate cell proliferation during murine palatogenesis.

Abnormal regulation of Sonic hedgehog (Shh) signaling has been described in a variety of human cancers and developmental anomalies, which highlights the essential role of this signaling molecule in cell cycle regulation and embryonic development. Gas1 and Boc are membrane co-receptors for Shh, which demonstrate overlapping domains of expression in the early face. This study aims to investigate potential interactions between these co-receptors during formation of the secondary palate. Mice with targeted mutation in Gas1 and Boc were used to generate Gas1; Boc compound mutants. The expression of key Hedgehog signaling family members was examined in detail during palatogenesis via radioactive in situ hybridization. Morphometric analysis involved computational quantification of BrdU-labeling and cell packing; whilst TUNEL staining was used to assay cell death. Ablation of Boc in a Gas1 mutant background leads to reduced Shh activity in the palatal shelves and an increase in the penetrance and severity of cleft palate, associated with failed elevation, increased proliferation and reduced cell death. Our findings suggest a dual requirement for Boc and Gas1 during early development of the palate, mediating cell cycle regulation during growth and subsequent fusion of the palatal shelves.

Paths of Heritable Mitochondrial DNA Mutation and Heteroplasmy in Reference and gas-1 Strains of Caenorhabditis elegans.

Heteroplasmy—the presence of more than one mitochondrial DNA (mtDNA) sequence type in a cell, tissue, or individual—impacts human mitochondrial disease and numerous aging-related syndromes. Understanding the trans-generational dynamics of mtDNA is critical to understanding the underlying mechanisms of mitochondrial disease and evolution. We investigated mtDNA mutation and heteroplasmy using a set of wild-type (N2 strain) and mitochondrial electron transport chain (ETC) mutant (gas-1) mutant Caenorhabditis elegans mutation-accumulation (MA) lines. The N2 MA lines, derived from a previous experiment, were bottlenecked for 250 generations. The gas-1 MA lines were created for this study, and bottlenecked in the laboratory for up to 50 generations. We applied Illumina-MiSeq DNA sequencing to L1 larvae from five gas-1 MA lines and five N2 MA lines to detect and characterize mtDNA mutation and heteroplasmic inheritance patterns evolving under extreme drift. mtDNA copy number increased in both sets of MA lines: three-fold on average among the gas-1 MA lines and five-fold on average among N2 MA lines. Eight heteroplasmic single base substitution polymorphisms were detected in the gas-1 MA lines; only one was observed in the N2 MA lines. Heteroplasmy frequencies ranged broadly in the gas-1 MA lines, from as low as 2.3% to complete fixation (homoplasmy). An initially low-frequency (<5%) heteroplasmy discovered in the gas-1 progenitor was observed to fix in one gas-1 MA line, achieve higher frequency (37.4%) in another, and be lost in the other three lines. A similar low-frequency heteroplasmy was detected in the N2 progenitor, but was lost in all five N2 MA lines. We identified three insertion-deletion (indel) heteroplasmies in gas-1 MA lines and six indel variants in the N2 MA lines, most occurring at homopolymeric nucleotide runs. The observed bias toward accumulation of single nucleotide polymorphisms in gas-1 MA lines is consistent with the idea that impaired mitochondrial activity renders mtDNA more vulnerable to this type of mutation. The consistent increases in mtDNA copy number implies that extreme genetic drift provides a permissive environment for elevated organelle genome copy number in C. elegans reference and gas-1 strains. This study broadens our understanding of the heteroplasmic mitochondrial mutation process in a multicellular model organism.

Growth Arrest Specific-1 (GAS1) Is a C/EBP Target Gene That Functions in Ovulation and Corpus Luteum Formation in Mice.

Ovulation and luteinization are initiated in preovulatory follicles by the luteinizing hormone (LH) surge; however, the signaling events that mediate LH actions in these follicles remain incompletely defined. Two key transcription factors that are targets of LH surge are C/EBPalpha and C/EBPbeta, and their depletion in granulosa cells results in complete infertility. Microarray analyses of these mutant mice revealed altered expression of a number of genes, including growth arrest specific-1 (Gas1). To investigate functions of Gas1 in ovulation- and luteinization-related processes, we crossed Cyp19a1-Cre and Gas1flox/flox mice to conditionally delete Gas1 in granulosa and cumulus cells. While expression of Gas1 is dramatically increased in granulosa and cumulus cells around 12–16 h post-human chorionic gonadotropin (hCG) stimulation in wild-type mice, this increase is abolished in Cebpa/b double mutant and in Gas1 mutant mice. GAS1 is also dynamically expressed in stromal cells of the ovary independent of C/EBPalpha/beta. Female Gas1 mutant mice are fertile, exhibit enhanced rates of ovulation, increased fertility, and higher levels of Areg and Lhcgr mRNA in granulosa cells. The morphological appearance and vascularization of corpora lutea appeared normal in these mutant females. Interestingly, levels of mRNA for a number of genes (Cyp11a1, Star, Wnt4, Prlr, Cd52, and Sema3a) associated with luteinization are decreased in corpora lutea of Gas1 mutant mice as compared with controls at 24 h post-hCG; these differences were no longer detectable by 48 h post-hCG. The C/EBP target Gas1 is induced in granulosa cells and is associated with ovulation and luteinization.

Role of Metabolic Shifts in Protection from Mutation Damage: Characterizing Mitochondrial Membrane Potential in C. Elegans Gas-1 Mutants

Many terminal human diseases are caused by mutations affecting mitochondrial functioning. Mitochondria are essential organelles responsible for producing cellular energy, adenosine triphosphate (ATP) via oxidative phosphorylation (OXPHOS) at mitochondrial electron transport chains (ETC). Proper ETC functioning relies on maintenance of the electrochemical gradient essential for energy production, known as mitochondrial membrane potential (∆ψM). The inner mitochondrial membrane is the site of the ETC and is most closely in contact with the enzymatic processes occurring within the mitochondrial matrix. Mutations affecting protein components of the ETC are especially troublesome for organelle health. ETC mutants commonly express altered ∆ψM, as well as increased production of damaging reactive oxygen species (ROS), which are hypothesized to cause genomic damage and lasting mutation. The nematode C. elegans is a practical model organism for investigating the phenotypic and genomic consequences of ETC mutations. Despite expressing higher levels of damaging ROS, the ETC protein complex 1 mutant, gas-1, expresses heritable mtDNA and nDNA mutation rates identical to those of wild type animals. I am using a mitochondria-targeted dye and fluorescence microscopy to quantify and compare ∆ψM levels of the gas-1 mutant and wild type animals. This work will provide a novel phenotypic characterization of this mutant and indicate whether decreased metabolic activity (e.g., reduced reliance on OXPHOS) is occurring in gas-1, and perhaps conferring protection from genomic degradation. Based on gas-1’s characteristically low ATP production and high ROS production, I expect that ∆ψM will be higher in gas-1 as compared to wild type levels.

Pharmacologic targeting of sirtuin and PPAR signaling improves longevity and mitochondrial physiology in respiratory chain complex I mutant Caenorhabditis elegans

Mitochondrial respiratory chain (RC) diseases are highly morbid multi-systemic conditions for which few effective therapies exist. Given the essential role of sirtuin and PPAR signaling in mediating both mitochondrial physiology and the cellular response to metabolic stress in RC complex I (CI) disease, we postulated that drugs that alter these signaling pathways either directly (resveratrol for sirtuin, rosiglitazone for PPARγ, fenofibrate for PPARα), or indirectly by increasing NAD+ availability (nicotinic acid), might offer effective treatment strategies for primary RC disease. Integrated effects of targeting these cellular signaling pathways on animal lifespan and multi-dimensional in vivo parameters were studied in gas-1(fc21) relative to wild-type (N2 Bristol) worms. Specifically, animal lifespan, transcriptome profiles, mitochondrial oxidant burden, mitochondrial membrane potential, mitochondrial content, amino acid profiles, stable isotope-based intermediary metabolic flux, and total nematode NADH and NAD+ concentrations were compared. Shortened gas-1(fc21) mutant lifespan was rescued with either resveratrol or nicotinic acid, regardless of whether treatments were begun at the early larval stage or in young adulthood. Rosiglitazone administration beginning in young adult stage animals also rescued lifespan. All drug treatments reversed the most significant transcriptome alterations at the biochemical pathway level relative to untreated gas-1(fc21) animals. Interestingly, increased mitochondrial oxidant burden in gas-1(fc21) was reduced with nicotinic acid but exacerbated significantly by resveratrol and modestly by fenofibrate, with little change by rosiglitazone treatment. In contrast, the reduced mitochondrial membrane potential of mutant worms was further decreased by nicotinic acid but restored by either resveratrol, rosiglitazone, or fenofibrate. Using a novel HPLC assay, we discovered that gas-1(fc21) worms have significant deficiencies of NAD+ and NADH. Whereas resveratrol restored concentrations of both metabolites, nicotinic acid only restored NADH. Characteristic branched chain amino acid elevations in gas-1(fc21) animals were normalized completely by nicotinic acid and largely by resveratrol, but not by either rosiglitazone or fenofibrate. We developed a visualization system to enable objective integration of these multi-faceted physiologic endpoints, an approach that will likely be useful to apply in future drug treatment studies in human patients with mitochondrial disease. Overall, these data demonstrate that direct or indirect pharmacologic restoration of altered sirtuin and PPAR signaling can yield significant health and longevity benefits, although by divergent bioenergetic mechanism(s), in a nematode model of mitochondrial RC complex I disease. Thus, these animal model studies introduce important, integrated insights that may ultimately yield rational treatment strategies for human RC disease.

In vivo metabolic flux profiling with stable isotopes discriminates sites and quantifies effects of mitochondrial dysfunction in C. elegans

Mitochondrial respiratory chain (RC) disease diagnosis is complicated both by an absence of biomarkers that sufficiently divulge all cases and limited capacity to quantify adverse effects across intermediary metabolism. We applied high performance liquid chromatography (HPLC) and mass spectrometry (MS) studies of stable-isotope based precursor–product relationships in the nematode, C. elegans, to interrogate in vivo differences in metabolic flux among distinct genetic models of primary RC defects and closely related metabolic disorders. MethodsC. elegans strains studied harbor single nuclear gene defects in complex I, II, or III RC subunits (gas-1, mev-1, isp-1); enzymes involved in coenzyme Q biosynthesis (clk-1), the tricarboxylic acid cycle (TCA, idh-1), or pyruvate metabolism (pdha-1); and central nodes of the nutrient-sensing signaling network that involve insulin response (daf-2) or the sirtuin homologue (sir-2.1). Synchronous populations of 2000 early larval stage worms were fed standard Escherichia coli on nematode growth media plates containing 1,6-13C2-glucose throughout their developmental period, with samples extracted on the first day of adult life in 4% perchloric acid with an internal standard. Quantitation of whole animal free amino acid concentrations and isotopic incorporation into amino and organic acids throughout development was performed in all strains by HPLC and isotope ratio MS, respectively. GC/MS analysis was also performed to quantify absolute isotopic incorporation in all molecular species of key TCA cycle intermediates in gas-1 and N2 adult worms. ResultsGenetic mutations within different metabolic pathways displayed distinct metabolic profiles. RC complex I (gas-1) and III (isp-1) subunit mutants, together with the coenzyme Q biosynthetic mutant (clk-1), shared a similar amino acid profile of elevated alanine and decreased glutamate. The metabolic signature of the complex II mutant (mev-1) was distinct from that of the other RC mutants but resembled that of the TCA cycle mutant (idh-1) and both signaling mutants (daf-2 and sir-2.1). All branched chain amino acid levels were significantly increased in the complex I and III mutants but decreased in the PDH mutant (pdha-1). The RC complex I, coenzyme Q, TCA cycle, and PDH mutants shared significantly increased relative enrichment of lactate+1 and absolute concentration of alanine+1, while glutamate+1 enrichment was significantly decreased uniquely in the RC mutants. Relative intermediary flux analyses were suggestive of proximal TCA cycle disruption in idh-1, completely reduced TCA cycle flux in sir-2.1, and apparent distal TCA cycle alteration in daf-2. GC/MS analysis with universally-labeled 13C-glucose in adult worms further showed significantly increased isotopic enrichment in lactate, citrate, and malate species in the complex I (gas-1) mutant. ConclusionsStable isotopic/mass spectrometric analysis can sensitively discriminate primary RC dysfunction from genetic deficiencies affecting either the TCA cycle or pyruvate metabolism. These data are further suggestive that metabolic flux analysis using stable isotopes may offer a robust means to discriminate and quantify the secondary effects of primary RC dysfunction across intermediary metabolism.

Succinate Dehydrogenase Upregulation Destabilize Complex I and Limits the Lifespan of gas-1 Mutant

Many Caenorhabditis elegans mutants with dysfunctional mitochondrial electron transport chain are surprisingly long lived. Both short-lived (gas-1(fc21)) and long-lived (nuo-6(qm200)) mutants of mitochondrial complex I have been identified. However, it is not clear what are the pathways determining the difference in longevity. We show that even in a short-lived gas-1(fc21) mutant, many longevity assurance pathways, shown to be important for lifespan prolongation in long-lived mutants, are active. Beside similar dependence on alternative metabolic pathways, short-lived gas-1(fc21) mutants and long-lived nuo-6(qm200) mutants also activate hypoxia-inducible factor –1α (HIF-1α) stress pathway and mitochondrial unfolded protein response (UPRmt). The major difference that we detected between mutants of different longevity, is in the massive loss of complex I accompanied by upregulation of complex II levels, only in short-lived, gas-1(fc21) mutant. We show that high levels of complex II negatively regulate longevity in gas-1(fc21) mutant by decreasing the stability of complex I. Furthermore, our results demonstrate that increase in complex I stability, improves mitochondrial function and decreases mitochondrial stress, putting it inside a “window” of mitochondrial dysfunction that allows lifespan prolongation.

Gastrointestinal defects of the Gas1 mutant involve dysregulated Hedgehog and Ret signaling

SummaryThe gastrointestinal (GI) tract defines the digestive system and is composed of the stomach, intestine and colon. Among the major cell types lining radially along the GI tract are the epithelium, mucosa, smooth muscles and enteric neurons. The Hedgehog (Hh) pathway has been implicated in directing various aspects of the developing GI tract, notably the mucosa and smooth muscle growth, and enteric neuron patterning, while the Ret signaling pathway is selectively required for enteric neuron migration, proliferation, and differentiation. The growth arrest specific gene 1 (Gas1) encodes a GPI-anchored membrane protein known to bind to Sonic Hh (Shh), Indian Hh (Ihh), and Ret. However, its role in the GI tract has not been examined. Here we show that the Gas1 mutant GI tract, compared to the control, is shorter, has thinner smooth muscles, and contains more enteric progenitors that are abnormally distributed. These phenotypes are similar to those of the Shh mutant, supporting that Gas1 mediates most of the Shh activity in the GI tract. Because Gas1 has been shown to inhibit Ret signaling elicited by Glial cell line-derived neurotrophic factor (Gdnf), we explored whether Gas1 mutant enteric neurons displayed any alteration of Ret signaling levels. Indeed, isolated mutant enteric progenitors not only showed increased levels of phospho-Ret and its downstream effectors, phospho-Akt and phospho-Erk, but also displayed altered responses to Gdnf and Shh. We therefore conclude that phenotypes observed in the Gas1 mutant are due to a combination of reduced Hh signaling and increased Ret signaling.

Gas1 Interferes with AβPP Trafficking by Facilitating the Accumulation of Immature AβPP in Endoplasmic Reticulum-Associated Raft Subdomains

The amyloid-β protein precursor (AβPP) is a type I transmembrane protein that undergoes maturation during trafficking in the secretory pathway. Proper maturation and trafficking of AβPP are necessary prerequisites for AβPP processing to generate amyloid-β (Aβ), the core component of Alzheimer's disease senile plaques. Recently, we reported that the glycosylphosphatidylinositol (GPI)-anchored protein growth arrest-specific 1 (Gas1) binds to and interferes with the maturation and processing of AβPP. Gas1 expression led to a trafficking blockade of AβPP between the endoplasmic reticulum (ER) and the Golgi. GPI-anchored proteins can exit the ER by transiting through raft subdomains acting as specialized sorting platforms. Here, we show that Gas1 co-partitioned and formed a complex with AβPP in raft fractions, wherein Gas1 overexpression triggered immature AβPP accumulation. Pharmacological interference of ER to Golgi transport increased immature AβPP accumulation upon Gas1 expression in these raft fractions, which were found to be positive for the COPII protein complex component Sec31A, a specific marker for ER exit sites. Furthermore, a Gas1 mutant lacking the GPI anchor that could not transit through rafts was still able to form a complex with AβPP but did not lead to immature AβPP accumulation in rafts. Together these data show that Gas1 interfered with AβPP trafficking by interacting with AβPP to facilitate its translocation into specialized ER-associated rafts where immature AβPP accumulated.

Overlapping Roles and Collective Requirement for the Coreceptors GAS1, CDO, and BOC in SHH Pathway Function

Secreted Hedgehog (HH) ligands signal through the canonical receptor Patched (PTCH1). However, recent studies implicate three additional HH-binding, cell-surface proteins, GAS1, CDO, and BOC, as putative coreceptors for HH ligands. A central question is to what degree these coreceptors function similarly and what their collective requirement in HH signal transduction is. Here we provide evidence that GAS1, CDO, and BOC play overlapping and essential roles during HH-mediated ventral neural patterning of the mammalian neural tube. Specifically, we demonstrate two important roles for these molecules: an early role in cell fate specification of multiple neural progenitors and a later role in motor neuron progenitor maintenance. Most strikingly, genetic loss-of-function experiments indicate an obligatory requirement for GAS1, CDO, and BOC in HH pathway activity in multiple tissues.

Assembly of the Yeast Cell Wall: Crh1p AND Crh2p ACT AS TRANSGLYCOSYLASES IN VIVO AND IN VITRO

The cross-linking of polysaccharides to assemble new cell wall in fungi requires mechanisms by which a preexisting linkage is broken for each new one made, to allow for the absence of free energy sources outside the plasma membrane. Previous work showed that Crh1p and Crh2p, putative transglycosylases, are required for the linkage of chitin to beta(1-3)glucose branches of beta(1-6)glucan in the cell wall of budding yeast. To explore the linking reaction in vivo and in vitro, we used fluorescent sulforhodamine-linked laminari-oligosaccharides as artificial chitin acceptors. In vivo, fluorescence was detected in bud scars and at a lower level in the cell contour, both being dependent on the CRH genes. The linking reaction was also shown in digitonin-permeabilized cells, with UDP-N-acetylglucosamine as the substrate for nascent chitin production. Both the nucleotide and the Crh proteins were required here. A gas1 mutant that overexpresses Crh1p showed very high fluorescence both in intact and permeabilized cells. In the latter, fluorescence was still incorporated in patches in the absence of UDP-GlcNAc. Isolated cell walls of this strain, when incubated with sulforhodamine-oligosaccharide, also showed Crhp-dependent fluorescence in patches, which were identified as bud scars. In all three systems, binding of the fluorescent material to chitin was verified by chitinase digestion. Moreover, the cell wall reaction was inhibited by chitooligosaccharides. These results demonstrate that the Crh proteins act by transferring chitin chains to beta(1-6)glucan, with a newly observed high activity in the bud scar. The importance of transglycosylation for cell wall assembly is thus firmly established.

Mitochondrial complex I function affects halothane sensitivity in Caenorhabditis elegans.

: The gene gas-1 encodes a subunit of complex I of the mitochondrial electron transport chain in Caenorhabditis elegans. A mutation in gas-1 profoundly increases sensitivity of C. elegans to volatile anesthetics. It is unclear which aspects of mitochondrial function account for the hypersensitivity of the mutant. : Oxidative phosphorylation was determined by measuring mitochondrial oxygen consumption using electron donors specific for either complex I or complex II. Adenosine triphosphate concentrations were determined by measuring luciferase activity. Oxidative damage to mitochondrial proteins was identified using specific antibodies. : Halothane inhibited oxidative phosphorylation in isolated wild-type mitochondria within a concentration range that immobilizes intact worms. At equal halothane concentrations, complex I activity but not complex II activity was lower in mitochondria from mutant (gas-1) animals than from wild-type (N2) animals. The halothane concentrations needed to immobilize 50% of N2 or gas-1 animals, respectively, did not reduce oxidative phosphorylation to identical rates in the two strains. In air, adenosine triphosphate concentrations were similar for N2 and gas-1 but were decreased in the presence of halothane only in gas-1 animals. Oxygen tension changed the sensitivity of both strains to halothane. When nematodes were raised in room air, oxidative damage to mitochondrial proteins was increased in the mutant animal compared with the wild type. : Rates of oxidative phosphorylation and changes in adenosine triphosphate concentrations by themselves do not control anesthetic-induced immobility of wild-type C. elegans. However, they may contribute to the increased sensitivity to volatile anesthetics of the gas-1 mutant. Oxidative damage to proteins may be an important contributor to sensitivity to volatile anesthetics in C. elegans.

Growth arrest specific gene 1acts as a region-specific mediator of theFgf10/Fgf8regulatory loop in the limb

Proximal-to-distal growth of the embryonic limbs requires Fgf10 in the mesenchyme to activate Fgf8 in the apical ectodermal ridge (AER), which in turn promotes mesenchymal outgrowth. We show here that the growth arrest specific gene 1 (Gas1) is required in the mesenchyme for the normal regulation of Fgf10/Fgf8. Gas1 mutant limbs have defects in the proliferation of the AER and the mesenchyme and develop with small autopods, missing phalanges and anterior digit syndactyly. At the molecular level, Fgf10 expression at the distal tip mesenchyme immediately underneath the AER is preferentially affected in the mutant limb, coinciding with the loss of Fgf8 expression in the AER. To test whether FGF10 deficiency is an underlying cause of the Gas1 mutant phenotype, we employed a limb culture system in conjunction with microinjection of recombinant proteins. In this system, FGF10 but not FGF8 protein injected into the mutant distal tip mesenchyme restores Fgf8 expression in the AER. Our data provide evidence that Gas1 acts to maintain high levels of FGF10 at the tip mesenchyme and support the proposal that Fgf10 expression in this region is crucial for maintaining Fgf8 expression in the AER.

Maintenance of cell integrity in the gas1 mutant of Saccharomyces cerevisiae requires the Chs3p-targeting and activation pathway and involves an unusual Chs3p localization.

Chitin synthase III is essential for the increase in chitin level and for cell integrity in cells lacking Gas1p, a beta(1,3)-glucanosyltransferase. In order to discover whether the upregulation of chitin synthesis proceeds through the canonical transport and activation pathway of Chs3p or through an alternative one, here we studied the effects of the inactivation of the GAS1 and CHS4-5-6-7 genes. All the double-null mutants showed a temperature-sensitive cell lysis phenotype that could be suppressed by the presence of an osmotic stabilizer. In liquid YEPD at 30 degrees C, chs4 delta gas1 delta, chs5 delta gas1 delta, chs6 deltagas1 delta and chs7 delta gas1 delta mutants were unable to grow, whereas they grew very slowly in minimal medium and showed low viability. High osmolarity suppressed the defective phenotype and restored growth. In chs4 gas1, chs5 gas1 and chs7 gas1, chitin levels did not increase and were reduced to only 10%, while in chs6 gas1 the value of gas1 was reduced to 20-40%. To investigate at which level the upregulation of chitin synthesis could occur, mRNA levels were monitored. The expression of CHS4-5-6-7 did not change significantly in gas1 delta. In strains expressing HA-tagged forms, the localization of Chs3p and Chs5p was examined. In the gas1 mutant the fluorescence pattern was affected and the proteins appeared abnormally present in the bud. The results indicate that: (a) the function of the CHS4-7 genes is required for chitin hyperaccumulation in gas1 mutant and for cell integrity; (b) homologous genes do not replace their function; (c) the regulation of CHS4-7 genes does not occur at transcriptional level. Control of the position of chitin synthesis could be important in protecting the bud from lysis.